CN101230394A - Multiplex amplification of short tandem repeat loci - Google Patents
Multiplex amplification of short tandem repeat loci Download PDFInfo
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Abstract
Methods and materials are disclosed for use in simultaneously amplifying at least eleven loci of genomic DNA in a single multiplex reaction, as are methods and materials for use in the analysis of the products of such reactions. Included in the present invention are materials and methods for the simultaneous amplification of at least eleven short tandem repeat loci, including specific materials and methods for the analysis of eleven such loci specifically selected by the United States Federal Bureau of Investigation as core loci for use in the Combined DNA Index System (CODIS) database.
Description
The application be that November 24, application number in 1999 are 99813729.4 the applying date, denomination of invention divides an application for the application for a patent for invention of " multiplex amplification of short tandem repeat ".
The cross reference of related application
The application is a U.S. Patent Application Serial 08/632,575 (April 15 1996 applying date), existing United States Patent (USP) 5,843, the part continuation application of 660 (authorizing day on December 1st, 1998), and 08/632,575 is again the part continuation application of the U.S. Patent Application Serial 08/316,544 of application on September 30th, 1994.The full text of these applications is incorporated herein for referencial use.
The research that federal government subsidizes or the statement of exploitation
Do not have
Invention field
The present invention relates to detect the genetic marker in the genome system.The present invention be more particularly directed to increase the simultaneously genetic loci of a plurality of different variforms of polymerase chain reaction or other amplification system, in a reaction, to measure the allelotrope that is contained in each locus in the multiplicated system.
Background of invention
Dna typing is generally used for paternity test, and is used for determining the pedigree of horse, dog and other animal farm crop.Dna typing also is generally used for differentiating blood, saliva, seminal fluid and maybe needs to identify other local other tissue of finding in human traces at locality of a crime.The existence that dna typing also is used for the success of clinical assays bone marrow transplantation or failure and special cancerous tissue whether.Dna typing relates to analyzes the allelotrope that has interested feature, is commonly referred to the genomic dna of " mark ".At present used typing by special be designed to detect and analyze known in colony with one or more regional length of at least two kinds of multi-form dna markers that manifest and/or the difference in the sequence.This kind length and/or sequence variations are called " polymorphism ".Any zone (i.e. " locus ") that the DNA of this variation wherein takes place is called " polymorphic locus ".Method of the present invention and material all are designed for a plurality of locus that detect DNA, some of them or all be polymorphic locus.
Abundant polymorphic genetic marker with regard to length or sequence is attempted to be used for to identify the identity application for a long time, as the tissue sample that is used for forensic analysis of paternity test and discriminating collection.This mark and analyze the announcement and the exploitation of the method for this mark was in that many years had been carried out the exploitation of several stages in the past.
The DNA variant mark of first discriminating is that simple base replaces, i.e. simple sequence polymorphism, and it is detected by the Southern hybridization analysis usually.For example, the reference of the discriminating of this mark that is designed for the DNA that uses radioactivity probe analysis digestion with restriction enzyme is described, referring to Southern, E.M. (1975), J.Mol.Biol.98 (3): 503-507; Schumm, etal (1988), American Journal of Human Geneties 42:143-159; And Wyman, A.and White, R. (1980) Proc.Natl.Acad.Sci.U.S.A.77:6754-6758.
S-generation mark is big or small variant, i.e. length polymorphism, especially " but series connection repeats parameter " (VNTR) mark (Nakamura Y.et al. (1987) .Science 235:1616-1622; And United States Patent (USP) 4,963,663, authorize (1990) such as White; United States Patent (USP) 5,411, (1995) such as White are authorized in 859,4,963,663 continuation) and " moonlet " mark (Jeffreys etal. (1985a), Nature 314:67-73; Jeffreys et al, (1985b) Nature 316:76-79; United States Patent (USP) 5,175,082, contriver Feffreys).VNTR and moonlet mark all contain the zone with the several identical sequences of series system multiple.Core tumor-necrosis factor glycoproteins length is 10-70 base, and short core tumor-necrosis factor glycoproteins is called " moonlet " to be repeated, and length repeats to be called VNTRs.Different Individual contains these repetitions of different quantities among the crowd.The VNTR mark replaces polymorphism than base usually and has higher polymorphism, presents on single genetic loci sometimes near 40 or more a plurality of allelotrope.But, still need restriction enzyme digestion and cumbersome approaches such as Southern hybridization analysis subsequently to detect and analyze most of this marks.
Ensuing progress comprises polymerase chain reaction (PCR) (United States Patent (USP) 4,683,203, Mullis, K.B.) technology and analysis VNTR locus (Kasai, K.et al. (1990) JournalForensic Science 35 (5): associating 1196-1200).The VNTR locus that can increase is found, and it can not need Southern to shift and detected.Amplified production separates by agarose or polyacrylamide gel, and by during increasing, mixing radioactivity or by detecting with silver or ethidium bromide poststaining.But PCR can only be used for the relatively little DNA section of reliable amplification, the promptly only reliable DNA section of amplification length under 3000 bases.(Ponce，M.& Micol，L.(1992)NAR 20(3)：623；Decorte R.et al.(1990)DNA CellBiol，9(6)：461-469)。As a result, only developed considerably less increased VNTRs.
In recent years, the polymorphic short series connection as genetic marker repeats the announcement of (STRs) and develops to have excited linkage map exploitation, the simplification of the discriminating of Disease-causing gene and evaluation and dna typing and the process of accuracy.Particularly, contain polymorphic dinucleotides and repeat (Litt and Luty (1989) Am J.Hum Genet 3 (4): 599～605; Tautz, D. (1989) NAR 17:6463-6471; Weber and May (1989) Am J Hum Genet 44:388-396; German patent DE 3,834 636 C2, contriver Tautz, D; United States Patent (USP) 5,582,979, applicant Weber L.), has STR (Edwards, A. etc., (1991) Am.J.Hum.Genet, the 49:746-756 of 3-4 Nucleotide repeating unit; Hammond, H.A, etc., (1994) Am.J.Hum.Genet.55:175-189; Fregeau C.J. and Fourney, R.M. (1993) BioTechniques15 (1): 100-119; Schumm, J.W. etc., (1994), the 4th human international symposium, 1993, the PP.177-178 of identifying; United States Patent (USP) 5,364,759, Caskey etc.; German patent DE 3834636C2, Tautz, D.) and have 5-7 Nucleotide repeating unit STR (referring to for example Edwards, A. etc. (1991) nucleic acids research 19:4971; Chen etc. (1993) genome 15 (3): 621-5; Harada etc., (1994) Am.J.Hum.Genet.55:175-189; Commings etc. (1995) genome 29 (2): 390-6; With the Am.J.Genet.57:619-628 of Utah marker development group (1995); With Jurka and Pethiyagoda (1995) molecular evolution magazine 40:120-126) the discovery and the exploitation of polymorphic mark, overcome the defective of many previous methods.The STR mark is generally short than VNTRs mark, and they are than be more suitable for amplification at most VNTR marks.
The str locus seat is similar to the VNTR locus that can increase, wherein at the allelotrope of the amplification of each this locus based on length variations and can be different.It has been generally acknowledged that the str locus seat is lower than VNTR locus in the polymorphism of each genes of individuals seat.Therefore, wish single amplified reaction with separate in increase and detect a plurality of STR system, so that the information of a plurality of locus to be provided simultaneously.The system that contains a plurality of locus is called multiplicated system, and has set forth many this systems that contain maximum 11 different str locus seats.For example referring to ACLM's journal (9-14 day in February, 1998), Schumm, James W. etc., p53, B88, document is the same; Gibson, Sandra D, etc., p53, B89, document is the same; Lazaruk, Katherine etc., p51 B83, document is the same; Sparkes.R etc., Int J Legal Med (1996) 109:186-194; Amp F1 STR Profiler
TMPcr amplification test kit service manual (1997), by Perkin-Elmer Corp, i-viii and 1-1 to 1-10 announce; AmpF1STR Profiler
TMPcr amplification test kit service manual (1997) is announced by Perkin-ElmerCorp.i-viii and 1-1 to 1-10; AmpF1 STR COfiler
TMPcr amplification test kit service manual (1998), by Perkin-Elmer Corp, i-iii and 1-1 to 1-10 announce; The 9th the international human Conference Papers collection (7-10 day in October, 1998) of identifying announced Staub, Rick W etc., Poster Abstract 15 by Promega Corp; The same, Willard, Jeanne M. etc., Poster Abstract 73; And the same, Walsh, P.Sean etc., Speaker Abstract for 8:50am-9:20am, Thursday on October 8th, 1998.
The scheme of amplification of STR locus can be designed to produce little product, and general length is 60-500 base pair (bp), and the allelotrope of each locus is less than 100bp at interval usually.This makes can carry out the electrophoretic analysis of a plurality of systems simultaneously on identical gel, perhaps carry out capillary electrophoresis analysis by well-designed PCR primer, so the allelic scope of not overlapping other system of the potential amplified production of all of each system.The design of these systems is subjected to single gel or kapillary separates the part restriction of a plurality of locus difficulty.This is because be that the fragments of different sizes in gel or the kapillary are particularly than due to the steric compression of long segment at the segmental device of the DNA isolation that those skilled in the art use always.
FBI (FBI) has set up and has preserved a kind of built-up type DNA index system (" CODIS "), and this is a dna typing database of information.Local, state and national judiciary use CODIS, the medical jurisprudence DNA evidence that can relatively collect with the DNA information in the database in the crime scene, other state-run Database Systems have proved these mechanisms with solving a kind of effective tool of violent crime (for example referring to Nigzgoda, Stephen, Cambridge Healthtech Institoute ' s Second Annual Conference on DNAForensics:Science, Evidence, and Future Prospects (Nov.17-18,1998), p1-21; Niezgoda, Stephen, Proceeding From The EighthInternational Symposium or Human Identification 1997, PromegaCorporation announces (1998), p48-49; Frazier, Rachel R.E. etc., ibid, p56-60; Niezgoda, S.J.Profiles in DNA 1 (3): 12-13; Werrett, D.J.and Sparkes, R.Speaker Abstracts:9
ThInternational Symposium onHuman Identification (Oct.7-10,1998) p5-6).As of late, only be that restriction fragment length polymorphism (" the RFLP ") data that derive from special VNTR locus analysis are considered to the nucleus in the database.FBI has differentiated 13 polymorphism str locus seats that comprise in the CODIS database recently.These 13 CODIS str locus seats are HUMCSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31 (Budowle, Bruce and Moretti, Tamyra, SpeakerAbstracts:9
ThInternational Symposium on Human Identification (Oct.7-10,1998) p7-8).VNTR and STR flag data all are kept in the CODIS database at present.(for example referring to Niezgoda, Stephen, Sencond Annual Conterenceon DNA Forensics, ibid).Until the present invention, can in single reaction and subsequent analysis, be still limited by the locus number of coamplification.Especially, still untapped material or the method that goes out to be used for 13 of multiplex amplifications or more a plurality of str locus seat and be used to differentiate 13 polymorphism str locus of CODIS database seat.
Material of the present invention and method are designed for multiple analysis all kinds DNA and comprise the strand of various different sourcess and the specific polymorphic locus of double-stranded DNA.The present invention is the obvious improvement to prior art, improves the DNA profile and has been used for linkage analysis, and the crime judicial adjudication, blood relationship test and other medical jurisprudence, ability to see things in their true light, accuracy and the flux of using differentiated in medical science and heredity.
Summary of the invention
Purpose of the present invention provides the series of genes seat that increases simultaneously and comprises that a plurality of different short series connection of polymorphism repeat the method and the material of (STR) locus in single multiple reaction, it is used in combination PCR or other amplification system and gel electrophoresis, capillary electrophoresis or other separation and detection method are with the allelic relative length of analyzing and contrast is increased in this multiple reaction of each locus.The method of the multiple analysis series of genes seat that the present invention discloses was not set forth in the prior art.The elaboration of the many primer sequences that also do not disclose about having hereinafter in the prior art, all these primers all show it is useful in the multiplex amplification of the series of genes seat that is disclosed.
Another order of the present invention provides the method that is specific to multiplex amplification, test kit and primer.
These and other purpose of the present invention is analyzed or is measured in each multiple reaction allelic method that each locus exists and material simultaneously and realizes by the present invention.Usually, method of the present invention comprises the steps: that (a) obtains at least one DNA sample to be analyzed, wherein this DNA sample have at least 13 can be by the locus of coamplification; (b) at least 13 locus of this DNA sample of coamplification; And (c) detect the material of amplification in the mode of the polymorphism that discloses system for use in carrying.
In one embodiment, the invention provides the allelic method that exists in the series of genes seat of measuring simultaneously from one or more DNA sample, comprising:
(a) obtain at least one DNA sample to be analyzed;
(b) the series of genes seat of selection DNA sample, it comprises the short tandem repeat of at least 13 energy coamplifications;
(c) this series of genes seat of coamplification in a multiplex amplification reaction, wherein reaction product is the allelic mixture that increases from each coamplification locus of this series;
(d) estimate the allelotrope that increases in the mixture, with determine in the DNA sample should each analysis of series the allelotrope that exists of locus.
At least 4 in these at least 13 short tandem repeats are preferably selected from next group locus: D3S1539 D4S2368, D5S818, D7S820, D9S930, D10S1239, D13S317, D14S118, D14S548, D14S562, D16S490, D16S539, D16S753, D17S1298, D17S1299, D19S253, D20S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13A01, HUMBFXIII, HUMLIPOL, HUMvWFA31.
In another embodiment of the present invention, the series of genes seat of in described method steps (b), selecting comprise 13 CODIS str locus seats (be D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX and HUMvWFA31), they with method of the present invention can by self or with other locus coamplification and analysis.
The present invention provides the test kit of the series of genes seat of a kind of while analyzing gene group DNA on the other hand, it comprises the Oligonucleolide primers of the series of genes seat of the genomic dna that coamplification is to be analyzed, wherein this serial genes seat comprises the short tandem repeat of at least 13 energy coamplification in identical multiple reaction, and wherein primer is arranged in one or more container.More preferably, this test kit comprises that coamplification human gene group DNA's the Oligonucleolide primers of a series of at least 13 locus is right, and this serial genes seat comprises D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31.
The present invention provides the primer sequence and the primer of amplification people DNA specific gene seat right on the other hand.Use primer of the present invention and primer to see following to carrying out the DNA multiple analysis.Primer of the present invention is applicable to method of the present invention, and wherein they can use evaluation procedure to help this method with the form of mark, and are as described below.
The present invention program has saved the multiple analysis contained time that locus spent, financial resources and material resources.Method of the present invention can be with single amplified reaction coamplification 13 or more a plurality of in a test tube, even many as 16 or more a plurality of locus, and not be used in independent each locus of amplification or less locus in each test tube.
The present invention is used in particular for forensic analysis, and blood relationship is measured, monitoring bone marrow transplantation, fields such as linkage mapping and detection of genetic and cancer.Method of the present invention obviously improves accuracy, thereby people can be with the DNA of its comparative preparation from the different samples of same individual.Accurately contrast or distinguish that the sample that contains minute quantity DNA particularly needs aspect forensic application, many there incrminating evidences (and evidence of innocence) depend on the dna typing analysis.
Scientist especially medicolegist's a plurality of polymorphic locuses of having had recognized the need to analyzing DNA for a long time is significant with the coupling of determining two DNA sample rooms statistically.(Presley, L.A. etc., human for the third time international symposium's collection of thesis 1992, the p245-269 (, delivering in 1993) of identifying by Promega Corp; Bever, R.A. etc., human for the second time international symposium's collection of thesis 1991, the p103-128 (delivering) of identifying by Promega Corp.1992).Yet until the present invention, people can't analyze 13 or more a plurality of str locus seat simultaneously in a reaction.For understanding the importance of this multiple ability, be necessary to understand some mathematics that dna typing is analyzed.
Illustrate, suppose that each str locus seat has the genotype (i.e. two allelic banding patterns) of 1/10 probability.In other words, suppose that the chance that two individualities of selecting at random have single STR matching type is 1/10.Yet if analyze two different str locus seats, the chance of coupling at random of these two systems is 1/100.If analyze 3 str locus seats, the matcher at random of each of these three systems can be 1/1000, and the like.So how the increase that is easy to understand very much the str locus seat number of being analyzed will reduce mates possibility at random among the general crowd, thereby improve type and the crime scene evidence of violating by the contrast suspicion of crime, accurately differentiate suspicion of crime criminal probability at the scene.Same reason, method of the present invention can also improve accurate discriminating paternity, correctly mate myeloid tissue, the remarkable result of exploitation linkage mapping research, and the possibility of detection heredopathia and cancer.
Others of the present invention, characteristics and advantage will be presented in the preferred embodiments of the invention and the accompanying drawing following carrying out.
The accompanying drawing summary
Figure 1A is the locus D3S1358 to human gene group DNA's sample, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, amplified production carries out the figure as a result that three fluorescence detects in the time of HUMTPOX and HUMvWFA31, and it detects with ABIPRISM 310 genetic analyzers in embodiment 1.
Figure 1B carries out the figure as a result that three fluorescence detects to the control sample of processing as the mode of Figure 1A, does not have genomic dna in the amplified reaction.
Fig. 2 A is the locus D3S1358 to human gene group DNA's sample, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475, S159, amplified production carries out the figure as a result that three fluorescence detects in the time of with Amelogenin, and it detects with ABI PRISM 310 genetic analyzers in embodiment 2.
Fig. 2 B carries out the figure as a result that three fluorescence detects to the control sample of processing as Fig. 2 A mode, does not have the genomic dna substrate in amplified reaction.
Fig. 3 A is the locus D3S1358 to human gene group DNA's sample, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475, amplified production in the time of S159 and Amelogenin, carry out the figure as a result that three fluorescence detects, it detects with ABI PRISM 377DNA sequenator in embodiment 3.
Fig. 3 B carries out the figure as a result that three fluorescence detects to the control sample of processing as Fig. 3 A mode, does not have the genomic dna substrate in amplified reaction.
Fig. 4 A and 4B are locus D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475, the fluoroscopic examination result's of amplified production Laser Printing figure in the time of S159 and Amelogenin, be to use the fluorescein passage (Fig. 4 A) and the carboxyl tetramethyl-rhodamine passage (Fig. 4 B) of HitachiFMBIO II fluorescent scanning instrument to detect, shown in embodiment 4.
Fig. 5 A and 5B are locus D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475, the fluoroscopic examination result's of amplified production Laser Printing figure in the time of S159 and Amelogenin, be to use the fluorescein passage (Fig. 5 A) and the carboxyl tetramethyl-rhodamine passage (Fig. 5 B) of HitachiFMBIO II fluorescent scanning instrument to detect, as described in embodiment 5.
Fig. 6 A and 6B are locus D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTHIO, HUMTPOX, HUMVWF31, C221, the fluoroscopic examination result's of amplified production Laser Printing figure in the time of S159 and Amelogenin, be to use the fluorescein passage (Fig. 6 A) and the carboxyl tetramethyl-rhodamine passage (Fig. 6 B) of HitachiFMBIO II fluorescent scanning instrument to detect, as described in embodiment 6.
Detailed Description Of The Invention
A, definition
Help scope and detailed content clear and the as one man following term of understanding to give a definition, to be used for setting forth and explaining the present invention:
" allele ladder ": the normal size mark that is formed by the allele that from locus, increases.
" allele ": the hereditary variation relevant with a DNA section namely occupies one of two or the multiple version of the dna sequence dna of homologous genes seat.
" biological chemical name ": used herein is the standard biological chemical name, and wherein nucleotide base is called as: adenine (A); Thymidine (T); Guanine (G); And cytimidine (C). Corresponding nucleotides for example is deoxyguanosine-5 '-triphosphoric acid (dGTP).
" dna polymorphism ": two or more different nucleotide sequence is present in the situation in the identical hybridization group together in the dna sequence dna.
" locus " or " genetic loci ": a special position on the chromosome. The allele of locus is positioned at the same loci of homologue.
" locus-specific primer ": under the used condition of amplification method, with the part of described locus or its complementary strand specific hybridization, and not with other dna sequence dna effectively hybridization at least for an allelic primer of locus.
" the pentanucleotide series connection repeats ": a subclass of the following stated STR polymorphism. Unless otherwise indicated, complete STR contained in term " the pentanucleotide series connection repeats ", and wherein recurring unit is 5 base sequences, and incomplete STR, and wherein at least one recurring unit is that 5 bases repeat.
" polymerase chain reaction " or " PCR ": a kind of technology, wherein by sex change, with primer annealing, and the copy number of the cyclic amplification target DNA sequence that extends with archaeal dna polymerase is to about 106Doubly or more times. It is described that the method for PCR amplification nucleic acid sees United States Patent (USP) 4683195 and 4683202, at this its described method incorporated into reference.
" polymorphism short tandem repeat ": the str locus seat of the following stated, wherein the repetitive sequence component number in the genomic DNA specific region (and clean sequence length) is changing between allele and between individuality.
" polymorphism information capacity " or " PIC ": the measured value (Botstein etc., 1980) of the polymorphism quantity that locus exists. The PIC value is between 0～1.0, and high value shows more much higher state property. This measured value is that the value that presents of heterozygosis measured value is little than other conventional measured value that uses usually. If mark is high informationalized (heterozygosis surpasses about 70%), then the difference between heterozygosis and the PIC is less.
" primer ": with single stranded oligonucleotide or dna fragmentation that the DNA chain of locus is hybridized, 3 ' of primer end can be used as the polymerization site of archaeal dna polymerase in this way.
" primer to ": two primers, comprise and at the primer 1 of the strand hybridization of dna sequence dna to be amplified one end, and with the primer 2 of the hybridization of the other end on dna sequence dna complementary strand to be amplified.
" primer sites ": the target DNA zone of primer hybridization.
" short tandem repeat " or " str locus seat ": contain shortly, length is the genomic DNA zone of the repetitive sequence element of 3-7 base-pairs. Term STR has also been contained a zone of genomic DNA, and wherein the sequence series connection more than one 3-7 base repeats or have the base of interleaving ground to repeat, and condition is that at least one sequence series connection repeats 2 times at least. At least each sequence that repeats in STR once is referred to herein as " recurring unit ".
Sequence with the str locus seat of materials and methods analysis of the present invention can be divided into two kinds of general types: perfect form and forme fruste. Term used herein " perfect form " STR refers to contain series connection and repeats at least double-stranded DNA zone of the recurring unit of single 3-7 base of secondary, such as (AAAAT)2 Term used herein " forme fruste " STR refers to that at least two series connection that contain complete recurring unit repeat, DNA zone with at least one repetition of incomplete recurring unit, the dna sequence dna that wherein forms incomplete recurring unit can get in the comfortable complete recurring unit sequence and lack, insert or replace 1,2,3 or 4 bases, for example (AAAAT)12(AAAAAT)
5
AAT(AAATT)
4 Each forme fruste STR sequence contains at least one perfect form STR sequence. Especially, no matter perfect form or forme fruste, each STR sequence comprises that series connection repeats at least one recurring unit's sequence of at least 2 times, and recurring unit's sequence can represent by following formula (1):
(A
wG
xT
yC
z)n (1)
A wherein, G, T and C represent the Nucleotide of any order; W, x, y and z represent the number of each Nucleotide in the sequence, scope between the 0-7 and the w+x+y+z sum between 3-7; N represents the series connection multiplicity of sequence and is at least 2.
The selection of B, multiple reaction composition
Method of the present invention relates to selects suitable series of genes seat, primer and amplification scheme are to produce the allelotrope of amplification from the locus of a plurality of coamplifications, preferably the locus size of these a plurality of coamplifications is not overlapping, more preferably is so that people can distinguish allelic mode mark from big or small eclipsed different genes seat.In addition, present method relates to the short tandem repeat of selecting with the compatible use of single amplification scheme.The special combination of locus as herein described is that this application is distinctive.The combination of locus can be vetoed owing to one of above two kinds of reasons, or because one or more locus of combination can not produce suitable product amount, or generation is not represented real allelic fragment and is vetoed in this reaction.
Except the above-mentioned combination that discloses, successful combination also can be by " repetition test " locus combination, by selecting primer to sequence, and produces to differentiate the balance that all locus that comprise wherein all can increase by regulating primer concentration.In case disclosed material of the present invention and method, those skilled in the art can infer the selection locus that is used for method of the present invention and test kit rightly, primer to the whole bag of tricks of amplification technique.All these methods all should be in claim scope of the present invention.
The particularly important part was the allelic magnitude range that produces the amplification of each locus of coamplification in the comfortable multiplex amplification reaction step during method of the present invention was implemented.In present analytical technology, can be most preferred less than the system that the fragment of 500 bases detects by amplification.
It is to select a series of locus that comprise at least 13 str locus seats that method of the present invention begins, these 13 locus can be in a multiplex amplification reaction coamplification.The present invention select locus and Oligonucleolide primers be used for method at multiplex amplification reaction amplification gene seat see following, and illustration in an embodiment.
C, utilize the polyad of three above locus of polyad exploitation of three locus
In many different technologies any all can be used for selecting to be used for series of genes seat of the present invention.A preferred technology of developing the useful locus series that is used for this analytical procedure sees following.In case work out the polyad that contains 3 str locus seats, it can be used as the core that produces the polyad that contains 3 above locus.Like this, can produce the new combination of three above locus of 3 locus that comprise beginning.For example, contain locus D7S820, the core polyad of D13S317 and D5S818 can be used for producing the polyad of deriving of following locus:
D16S539, D7S820, D13S317 and D5S818;
HUMCSF1PO, HUMTPOX, D16S539, D7S820, D13S317 and D5S818;
HUMCSF1PO, HUMTPOX, HUMTH01, D16S539, D7S820, D13S317, and D5S818;
HUMCSF1PO, HUMTPOX, HUMTH01, HUMvWFA31, D16S539, D7S820, D13S317 and D5S818;
D3S1358, D5S818, D7S820, D8S1179, D13S317, D17S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMVWA31;
S159, HUMCSF1PO, D16S539, D7S820, D13S317 and D5S818;
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMSF1PO, HUMFIBRA, HUMTH01, HUMTPOX and HUMvWFA31;
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, HUMvWFA31, G475, S159 and Amelogenin.
Use method of the present invention, the locus of core series can be used for producing other suitable deutero-str locus seat series and is used for multiple analysis.No matter how to select the locus with methods analyst of the present invention, the locus that is used for multiple analysis of all selections all shows following feature: they produce enough amplified productions to estimate (1); (2) their produce seldom (if any) because in the illusion that adds during the multiplex amplification step due to (or not adding) base in the allelotrope of amplification; (3) their produce seldom (if any) because the illusion due to the polysaccharase premature termination amplified reaction; And (4) they seldom or not be created in the single base of consecutive miss under the allelotrope of given true amplification and less " hangover " of molecular weight that produce is with.For example referring to Schumm etc., the 4th the international human Conference Papers collection 1993 of differentiating, p177-187 (announcing) by Promega CorP.1994.
Differentiate the constructed of at least 3 locus as above-mentioned being used to,, can be used for selecting 13 or the locus or the multiple analysis of more a plurality of human genomes according to the preferred embodiment of analytical procedure of the present invention.Locus as any series of above-mentioned discriminating is applicable to multiple analysis of the present invention, and condition is that locus series comprises at least 13 str locus seats.More preferably, at least 4 locus of at least 13 str locus seats analyzing according to the present invention are selected from next group: D3S1539, D4S2368, D5S818, D7S820, D9S930, D10S1239, D13S317, D14S118, D14S548, D14S562, D16S490, D16S539, D16S753, D17S1298, D17S1299, D19S253, D20S481, D22S683, HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13A01, HUMBFXIII, HUMLIPOL, and HUMvWFA31.More preferably, the locus series of analyzing according to the present invention comprises whole 13 CODIS locus, i.e. D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA, HUMTH01, HUMTPOX, and HUMvWFA31.
At least one locus that selection is used for carrying out at this multiple reaction the locus of coamplification preferably has the str locus seat that length is the repeating unit of 5-7 base or base pair, and five Nucleotide multiple str locus seats are more preferably arranged.As incorporate into as described in the U.S. Patent application 09/018584 of reference, amplification has this moderate multiple locus minimum illusion takes place, and this illusion is for example because due to repeating to slide.The above locus series comprises that 3 have this locus of pentanucleotide multiple: G475, C221 and S159.At this used term " G475 ", " C221 " and " S159 " refers to the title as the U.S. Patent application 09/018584 described pentanucleotide duplicate loci of incorporating reference into.The clone of each pentanucleotide locus that each title is equivalent to therefrom differentiate.The sequence that is described as the G475 clone of SE ID NO:34 in this patent application is referred to herein as SEQ ID NO:108.The sequence that is described as the C221 clone of SEQ ID NO:2 in this article is referred to herein as SEQ ID NO:109.The sequence that is described as the S159 clone of SEQ ID NO:26 in this article is referred to herein as SEQ ID NO:110.Be used to the G475 that increases in this article, each primer of C221 and S159 and primer are to the homologous genes seat at least 13 locus of also can be used for increasing according to the present invention coamplification and analysis.
The locus series of selecting that is used for coamplification of the present invention and analysis preferably also comprises at least one locus except that these at least 13 str locus seats.This extra locus preferably includes sequence polymorphism, or differentiates and can differentiate the another kind of feature of the particular feature of body DNA one by one from other DNA of individual of crowd.This extra locus is the locus of the DNA sample source sex of discriminatory analysis more preferably.When this DNA sample was the human gene group DNA, the locus of discriminating source sex such as Amelogenin locus were to carry out coamplification of the present invention and analyze institute preferably.This Amelogenin locus differentiates in GenBank for HUMAMELY (when being used for identification packets and being contained on the male dna Y chromosome locus), or differentiate for HUMAMELX (when be used to differentiate male or the female dna X chromosome on during locus).When the Amelogenin locus in identical multiplex amplification reaction when at least 13 short tandem repeat coamplifications, in multiplex amplification reaction, be used to increase this special gene seat primer at least one have and be selected from following sequence: SEQ ID NO:86, SEQ IDNO:105 and SEQ ID NO:87.
D. select primer
In case differentiated the locus series of coamplification in a multiple reaction, people can determine to be suitable for the primer of each locus of coamplification.Care should be used to selects to be used for the primer sequence of multiple reaction.Incorrect selection primer can produce some undesired effects as not increasing, in the amplification of a plurality of sites, primer dimer forms, undesirable interaction of the primer sequence of different genes seat, the allelotrope that the allelotrope of a locus and another locus produce is overlapping, or need be in multiple reaction the amplification condition or the scheme of inconsistent different genes seat.The primer that is used for the primer of the inventive method or is included in test kit of the present invention is preferably selected according to following system of selection.
The primer that is used for multiplicated system of the present invention is mixed for the primer of the selected locus of coamplification preferably by repeating to select primer sequence, the coamplification locus, separate then and detect the amplification this process of product select.Beginning, this method produces the allelotrope (being the output height of some locus than other locus) of amplification usually in uneven mode, but also may produce the amplified production of not representing allelotrope self.These extra fragments can be by due to above-mentioned arbitrary reason.
For eliminate this extra fragment in the multiple analysis system, each primer uses together with the primer of identical or other locus, causes the amplification of extra fragments to differentiate which primer.In case differentiate to produce one or more this segmental two primers, with one of them or two primers all make an amendment and in pairs or the examination of resurveying in multiple analysis system (or part of multiple analysis system).But repeat this method does not have or only have receiving amount in the allelotrope output that increases in the multiple analysis system extra fragments.
Sometimes extra fragments can be by removing at the opposite primer of primer centering mark.This changes the product that shows phase anti-primer in the detection step.The primer of this new mark can increase than the true allelotrope of hi-fi than the primer of previous mark, produces the true allelotrope that accounts for total amplified production major part.
Determining and can carrying out before or after the final primer sequence of selection of primer concentration preferably carried out after selecting.Usually, the primer concentration that improves any special gene seat increases the product amount that this locus produces.Yet this also is a revision test process, because the increase of a locus output can reduce the output of one or more other locus.In addition, primer can interact and directly influence the output of other locus.The linearity that the linearity increase of primer concentration not necessarily produces corresponding gene seat output improves.
Balance between the locus also is subjected to the parameter influence of many amplification schemes, as template used quantity, and the amplification cycles number of times, the annealing temperature of thermal cycling scheme, and when working cycle finishes, comprise or do not comprise extra extension step.Complete equipilibrium between all allelotrope and locus does not reach usually.
The process of multiple analysis system development can also be the repetitive process of above-mentioned other principle.Promptly at first develop the multiple analysis system of a small amount of locus, this system does not have or does not almost have extra fragment in amplification.The primer of this system can with the combination of primers of one or more other locus.The combination of primers of this expansion may produce or not produce extra fragment in amplification, thereby can import and estimate new primer.
Repeat above-mentioned-or a plurality of repetition chosen process, until a whole set of primer that identifies at least 13 locus that carry out coamplification that can be used for coamplification such as above-mentioned selection.Ying Zhike develops the locus of the primer of many different series with the amplification particular series.
The synthetic primer that is used for the inventive method can use any oligonucleotide synthetic standards step well known by persons skilled in the art to carry out.At least one primer of each locus is preferably with the dye marker covalent attachment, as with as described in the F of lower section.
Following table 1 provides the primer sequence table of determining to be applicable to the corresponding polymorphism str locus seat that amplification is listed.At least one locus that at least one primer is preferred for increasing and carries out coamplification and analysis and select as above-mentioned in the table 1.Ying Zhike differentiates other primer of the following locus that is suitable for increasing simultaneously.
Table 1
| Locus | Primer SEQ ID NO: |
| |
1,2,80 and 81 |
| |
3,4,82 and 83 |
| D5S818 | 5,6,84 and 85 |
| D3S1539 | 7,8 and 49 |
| D17S1298 | 9 and 10 |
| |
11,12,52 and 53 |
| |
13,14,55 and 61 |
| |
15,16 and 54 |
| D14S118 | 17 and 18 |
| D14S562 | 19 and 20 |
| D14S548 | 21 and 22 |
| D16S490 | 23 and 24 |
| D16S753 | 25 and 26 |
| D17S1299 | 27 and 28 |
| D16S539 | 29,30,58,79 and 97 |
| D22S683 | 31 and 32 |
| HUMCSF1PO | 33,34,77,78 and 98 |
| HUMTPOX | 35,36,72 and 73 |
| HUMTH01 | 37,38,66,67 and 103 |
| HUMvWFA31 | 39,40,59,60 and 76 |
| Locus | Primer SEQ ID NO: |
| HUMF13A01 | 41 and 42 |
| HUMFESFPS | 43 and 44 |
| HUMBFXIII | 45 and 46 |
| HUMLIPOL | 47 and 48 |
| D19S253 | 50 and 51 |
| D4S2368 | 56 and 57 |
| D18S51 | 62,63,101 and 102 |
| D21S11 | 64 and 65 |
| D3S1538 | 68,69 and 106 |
| HUMFIBRA | 70,71 and 107 |
| D8S1179 | 74,75 and 104 |
| G475 | 88,89 and 94 |
| S159 | 90,91,92,93,95 and 96 |
| |
99 and 100 |
E. prepare the DNA sample
The genome DNA sample that is used for the inventive method can use the method for any the prepare DNA compatible with DNA amplification.The known many this methods of those skilled in the art.For example comprise but non-being limited to through phenol extraction purify DNA (Sambrook, (1989) molecular cloning laboratory manual such as J., second edition, cold spring harbor laboratory publishes, the cold spring port, New York, p9.14-9.19), through salt precipitation partial purification (Miller, S. etc. (1988) nucleic acids research 16:1215), or resin partial purification (Walsh etc., (1991) biotechnology 10:506-513, Comey etc., (1994) medical jurisprudence 39:1254), with discharge unpurified material (Burckhardt with untreated blood, J. (1994) PCR method and use 3:239-243, McCabe, Edward R.B. (1991) PCR method and use 1:99-106, Nordvag, Bjorn-Yngvar (1992) biotechnology 12:4 p490-492).
When at least one the DNA sample with methods analyst of the present invention is the human gene group DNA, this DNA preferred preparation is from being selected from following tissue: blood, seminal fluid, vaginal cell, hair, saliva, urine, bone, cheek sample, the amniotic fluid that contains placenta cells or fetal cell, Chorionic villi, and the above mixture that draws any tissue.
Preferably, DNA concentration can be measured with standard DNA quantivative approach well known by persons skilled in the art before being used for the inventive method.In this case, DNA concentration is preferably passed through Sambrook, J. etc. (1989), and as preceding, the E.5 described spectrophotometry of appendix is measured, or by (1979) such as Brunk C.F., the described fluorometry of analytical biochemistry 92:497-500 is measured.DNA concentration is more preferably measured by the quantity of contrast personnel selection specific probe hybrid dna standard substance, as Waye, J.S. etc. (1991) " sensitive and quantitative specifically people's gene group thymus nucleic acid (DNA) report of survey in the medical jurisprudence sample ", Journal of Forensic Sciences, 36:1198-1203 is described.In amplified reaction, use too many template DNA to produce and be rendered as the illusion of not representing true allelic extra band.
F. DNA amplification
In case prepared genome DNA sample, the target gene seat can be in the multiplex amplification step of the inventive method coamplification.In the different amplification methods any all can be used for the amplification gene seat, comprise but non-polymerase chain reaction (the PCR) (Saiki that is limited to, R.K. etc. (1985), science 230:1350-1354), based on the amplification (Kwoh that transcribes, D.Y. and Kwoh, T.J. (1990) U.S. Biotechnology Experiment chamber, October nineteen ninety), and strand displacement amplification (SDA) (Waller, G.T. etc. (1992), institute of American Academy of Sciences reports 89:392-396).Preferably, the DNA sample is with being specific to the primer of each locus to carrying out pcr amplification.The used primer sequence of following examples has at length been listed in appended sequence table in this specification sheets back, and the some of them sequence is other embodiment of the present invention.
At least one primer of each locus preferably with the dye marker covalent attachment, more preferably with the fluorochrome label covalent attachment.Primer and accompanying dyestuff preferably select to be used for multiplex amplification reaction, so when allelotrope preferably passes through gel or capillary electrophoresis separation, with a kind of allelotrope of primer amplification of each locus of color mark, the allelotrope of other locus of not overlapping primer coamplification with the same color mark.
In particularly in the embodiment preferred of the inventive method, at least one primer of each locus of coamplification in multiple reaction was with the fluorescent mark mark before being used to react.It is commercially available to be suitable for adhering to the fluorescent mark that is used for primer of the present invention.For example available from fluorescein and the carboxyl tetramethyl-rhodamine mark and their derivative of PE biosystem and molecular probe company.More preferably, at least 3 different marks are used for the used different primers of mark multiplex amplification reaction.When estimating multiple reaction with big tick marks, the primer that is used for preparing big tick marks is preferably used the not isolabeling mark with the primer that is used for amplified reaction corresponding gene seat.
See for details in following examples about the most preferred amplification scheme that is used for every kind of most preferred locus combination of the inventive method.Embodiment has also described the special step relevant with each polyad in addition in detail.The sequence that is used for the locus-specific primer of embodiment comprises many Nucleotide, and these Nucleotide are hybridized with the allelotrope of the locus that increases under the used condition of hybridization fully, and the allelotrope of other locus that do not increase substantially.Referring to the United States Patent (USP) 5,192,668 of the Simons that incorporates reference into, it has more elaborated the locus-specific primer.
G. prepare and detect dna fragmentation
In case the multiplex amplification step through the inventive method has produced the allelotrope of a series of amplifications, the allelotrope of amplification is estimated.The evaluation procedure of this method can be undertaken by any different methods, preferred the following stated method.
Preferably use the product of electrophoretic separation multiplex amplification reaction, more preferably capillary electrophoresis is (for example referring to Buel, Eric etc., (1998), Journal of Forensic Sciences 43 (1) p164-170), or denaturing polyacrylamide gel electrophoresis is (for example referring to Sambrook J. etc., (1989), molecular cloning laboratory manual, second edition, cold spring harbor laboratory publishes, p13.45-13.57).Following examples illustration be applicable to preparing gel and the electrophoresis step and the condition of the inventive method evaluation procedure.The DNA isolation fragment is mainly based on clip size in denaturing polyacrylamide gel electricity arteries and veins and capillary electrophoresis.
In case separated the allelotrope of amplification, can observe and analyze to the allelotrope in gel or the kapillary and any other DNA (as big tick marks of DNA or allelotrope ladder).Observation DNA can carry out with any prior art in gel, comprises that silver dyes or reporter such as radio isotope, fluorophore, chemiluminescent substance and enzyme with can detect substrate and make up.Yet, detect contain 13 or the preferred method of the polyad of more a plurality of locus be fluorometric assay (referring to Schumm, J.W. etc., the 8th the international human Conference Papers collection of identifying, (announcing), p78-84 by PromegaCorporation; Buel, Eric etc., (1998) are as preceding)), wherein the primer of each locus is followed the trail of with the product of fluorescence detector certification mark in the multiple reaction.Above-mentioned elaboration observes allelic existing method incorporate reference at this.
The allelotrope that exists in the DNA sample is preferably by comparing with size criteria thing such as dna marker or locus-specific allelotrope ladder, with the allelotrope of determining that each locus exists in the sample.Evaluation contain two or the most preferred size criteria thing of the multiplex amplification of more a plurality of polymorphism str locus seats form by the allelotrope ladder of each locus of being estimated.Referring to Puers, Christoph etc., (1993) Am J.Hum Genet.53:953-958, Puers.Christoph etc., (1994) Genomics 23:260-264.Referring to United States Patent (USP) 5599666,5674686 and 5783406, these patents have been set forth the allelotrope ladder that is applicable in the detection of str locus seat in addition, and have disclosed the construction process of allelotrope ladder.
After making up the allelotrope ladder of each locus, they are mixed carry out gel electrophoresis when being incorporated in the amplification sample.The allelotrope of each allelotrope ladder corresponding gene seat in sample moves.
The product of multiple reaction of the present invention can be estimated with inner swimming lane standard substance, and this standard substance is the size criteria thing that is designed to electrophoretic a kind of special type in polyacrylamide gel or identical identical swimming lane capillaceous.This inner swimming lane standard substance preferably is made up of the fragment of a series of known length.This inner swimming lane standard substance more preferably is with the fluorochrome label that can separate with other dye area in the amplified reaction.
After making up inner swimming lane standard substance, this standard substance also can mix with the sample or the allelotrope ladder of amplification, and carries out electrophoresis with the migration in the different kapillaries of the different swimming lanes that contrast gel electrophoresis or capillary electrophoresis.The variation of inner swimming lane standard substance migration shows the separating medium changes of properties.These differences quantitatively and with the allelotrope ladder are proofreaied and correct, to proofread and correct allelic size measurement in the unknown sample.
H, preferred detection technique: fluoroscopic examination
In the most preferred embodiment of the inventive method, fluoroscopic examination is used for estimating the allelotrope that mixture that multiplex amplification reaction produces increases.Below briefly set forth and how to have used preferred detection method.
Along with the appearance of automatic fluorescent imaging, can rapid detection and analyze the multiplex amplification product.For fluorometric analysis, can comprise a fluorescently-labeled primer in the amplification of each locus.Be applicable to that preferably fluorescently-labeled primer of the present invention comprises (TMR-) and 5 of fluorescein-labeled (FL-), carboxyl tetramethyl-rhodamine mark, (R6G) primer of 6-carboxyl rhodamine 6G mark is as institute's illustration in following examples.Preferably by plate gel electrophoresis or capillary electrophoresis, separate fragment with the amplification of the primer generation of this mark.The isolating fragment of gained can be used fluorescence detection device such as ABI PRISM 310 genetic analyzers, ABIPRISM 377 dna sequencing instrument (Applied Biosystems Division, PerkinElmer, Foster Crty, CA) or Hitachi FMBIO II fluorescent scanning instrument HitachiSoftware Engineering America, Ltd.South San Francisco CA) analyzes.
In brief, method of the present invention most preferably uses fluoroscopic examination as detecting step.In this preferred detection method, being used for one of every pair of primer of multiplex amplification reaction or two all has the fluorescent mark that is attached to it, thereby the allelotrope that produces from the amplification of amplified reaction is fluorescently-labeled.In this most preferred embodiment of the present invention, the allelotrope of amplification is subsequently by capillary electrophoresis separation, and observes and the allelotrope of analytical separation with the fluorescent imaging analyser.
Fluoroscopic examination specific activity mark and detection method are preferred, because it does not need to use radioactive substance, have avoided all laws and the safety problem of using this material to follow.
Use the fluoroscopic examination of labeled primer and also dye preferably, because fluorescence detection lacks than the illusion that silver dyeing detection method produces than other on-radiation detection method such as silver.Illusion is because the DNA cloning chain that just adheres to mark that detects in fluoroscopic examination than small part, and detects with silver dyeing detection method, and each allelic two chain that increase that then produces from the DNA of multiplex amplification reaction all are painted.
I, test kit
The invention still further relates to the test kit that utilizes aforesaid method.A basic test kit comprises a container with one or more locus-specific primer.Randomly can comprise operation instruction.
Other optional test kit composition can comprise the allelotrope ladder at each specific gene seat, the enzyme that is used to increase of q.s, promote the amplification buffer of amplification, the application of sample solution of electrophoretic amplified material is carried out in preparation, the genomic dna of template in contrast, determine material size criteria thing as expecting migration in isolation medium, reach the scheme and the handbook of instructing the user and limiting error in the use.The quantity of all ingredients also can change according to many factors in the test kit, the best susceptibility of these factors such as method.The present invention also provides the used test kit of manual operation or automatic tester or the used test kit of analyser.
Embodiment
Following examples are for example understood advantage of the present invention, and help those of skill in the art to produce and use.This embodiment just illustrates but not limits the protection that claim scope of the present invention or patent are granted by any way.
The human gene group DNA who analyzes in following examples prepares autoblood or tissue culture cells, uses the described standard step preparation of Miller and Dykes (Miller, S.Deng (1988), nucleic acids research 16:1215).Known usually in the described separation of this article and quantivative approach those skilled in the art, and be preferred during the present invention uses, but not necessarily.
Below each embodiment be a use-case of the inventive method, the allelotrope that exists at least 13 locus with one or more DNA sample of determining the human gene group DNA simultaneously.The locus series of each following coamplification comprises that 13 short tandem repeats that are used for the CODIS system of discriminating (are D3S1358, HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D3S317, D7S820, D16S539 and HUMCSF1PO.The locus series of some following coamplifications also comprises one or more extra short tandem repeat if any pentanucleotide multiple locus (for example G475, S159, or C221), and non-str locus seat such as Amelogenin.
Table 2 has been summarized described in following each embodiment the locus series of coamplification in the multiplex amplification reaction.This table has shown that also the primer of each this locus that is used to increase is right in each this multiple reaction.A right primer of listed each primer of table 2 was used fluorescent mark before being used for multiplex amplification reaction.In some cases, different marks is used for the primer of mark different genes seat, and so the allelotrope that produces with different primers when the fluorescence equipment with laser active detects, can be distinguished mutually.
Have three kinds of different fluorescent marks to be used for following examples, in the following table " FL " representative fluorescein-labeled, " TMR " representative is at carboxyl tetramethyl-rhodamine mark, " R6G " represents 5,6-carboxyl rhodamine 6G mark.Which primer that table 2 has also shown the every pair of primer that is used for multiplex amplification reaction each embodiment be mark like this (for example " FL-69 " mean the primer with SEQ ID NO:69 be used for multiplex amplification reaction before at its 5 ' end with fluorescein-labeled).Yet in the text of each embodiment, mark abbreviation places (for example " FL-SEQ ID NO:2 " replacement " FL-2 ") before the SEQ ID NO of primer of the used mark of described amplified reaction.
Table 2
| Embodiment | The locus of amplification | The primer is right: SEQ ID NO | Used |
| 1 | D3S1358 HUMTH01 D21S11 D18S51 HUMvWFA31 D8S1179 HUMTPOX HUMFIBRA D5S818 D13S317 D7S820 D16S539 HUMCSF1PO | 68，69 66，67 64，65 62，63 76，40 74，75 72，73 70，71 84，85 82，83 80，81 29，79 77，78 | FL-69 FL-66 FL-65 FL-62 TMR-40 TMR-75 TMR-73 TMR-70 R6G-85 R6G-83 R6G-80 R6G-79 R6G-78 |
| Embodiment | The locus of amplification | The primer is right: SEQ ID NO | Used |
| 2，3 | D3S1358 HUMTH01 D21S11 D18S51 G475 Amelogenin HUMvWFA31 D8S1179 HUMTPOX HUMFIBRA D5S818 D13S317 D7S820 D16S539 HUMCSF1PO S159 | 68，69 66，67 64，65 62，63 88，89 86，87 76，40 74，75 72，73 70，71 84，85 82，83 80，81 29，79 77，78 90，91 | FL-69 FL-66 FL-65 FL-62 FL-88 TMR-86 TMR-40 TMR-75 TMR-73 TMR-70 R6G-85 R6G-83 R6G-80 R6G-79 R6G-78 R6G-91 |
| 4 | D3S1358 HUMTH01 D21S11 D18S51 G475 Amelogenin HUMvWFA31 D8S1179 HUMTPOX HUMFIBRA D5S818 D13S317 D7S820 D16S539 HUMCSF1PO S159 | 68，69 66，67 64，65 62，63 88，89 86，87 76，40 74，75 72，73 70，71 84，85 82，83 80，81 29，79 77，78 90，91 | FL-69 FL-66 FL-65 FL-62 FL-88 TMR-86 TMR-40 TMR-75 TMR-73 TMR-70 FL-85 FL-83 FL-80 FL-79 FL-78 FL-91 |
| Embodiment | The locus of amplification | The primer is right: SEQ ID NO | Used fluorescent mark |
| 5 | D3S1358 HUMTH01 D21S11 D18S51 G475 Amelogenin HUMvWFA31 D8S1179 HUMTPOX HUMFIBRA D5S818 D13S317 D7S820 D16S539 HUMCSF1PO S159 | 68，69 66，67 64，65 62，63 88，94 86，87 76，40 74，75 72，73 70，71 84，85 82，83 80，81 29，79 77，78 95，96 | FL-69 FL-66 FL-65 FL-62 FL-94 TMR-86 TMR-40 TMR-75 TMR-73 TMR-70 FL-85 FL-83 FL-80 FL-79 FL-78 FL-96 |
| 6 | D3S1358 HUMTH01 D21S11 D18S51 S159 Amelogenin HUMvWFA31 D8S1179 HUMTPOX HUMFIBRA D5S818 D13S317 D7S820 D16S539 HUMCSF1PO C221 | 69，106 38，103 64，65 101，102 92，93 105，87 76，40 104，75 72，73 70，107 84，85 3，4 80，81 29，97 77，98 99，100 | FL-69 FL-38 FL-65 FL-101 FL-93 TMR-105 TMR-40 TMR-104 TMR-72 TMR-70 FL-85 FL-4 FL-80 FL-29 FL-98 FL-99 |
With ABI PRISM 310 genetic analyzer fluoroscopic examination locus D3S1358,
HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179, HUMTPOX,
HUMFIBRA, D5S818, D7S820, D13S317, D16S539, and HUMCSF1PO
Multiplex amplification
In this embodiment, each locus D3S1358 of the dna profiling that in a reaction tube, increases simultaneously, HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, and HUMCSF1PO.Pcr amplification is at 25 μ l, 1 * Gold ST*R damping fluid (50mM KCl, 10mM Tris-HCl (pH8.3 is at 25 ℃), 0.1%Triton X-100,1.5mM MgCl
2, 160 μ g/ml BSA and every kind of dATP of 200 μ M, dCTP, dGTP, and dTTP) in, with 1ng template and 3.25U AmpliTaq Gold
TMArchaeal dna polymerase carries out.(Perkin Elmer, Poster City use following amplification scheme in CA): 96 ℃ 12 minutes in GeneAmp PCR system 9600; Carried out 94 ℃ of 10 round-robin then 30 seconds, and in 68 seconds,, kept 30 seconds, in 50 seconds,, kept 45 seconds gradually to 70 ℃ gradually to 58 ℃; Carry out subsequently 20 round-robin 90 ℃ 30 seconds, in 60 seconds,, kept 30 seconds gradually to 58 ℃, in 50 seconds,, kept 45 seconds gradually to 70 ℃; Then carry out 1 time 60 ℃ 30 minutes.
Be used in combination 26 amplimers, comprise each 0.12 μ M D3S1358 primer 1 (SEQID NO:68) and primer 2 (FL-SEQ ID NO:69), each 0.08 μ M HUMTH01 primer 1 (FL-SEQ ID NO:66) and primer 2 (SEQ ID NO:67), each 0.8 μ M D21S11 primer 1 (SEQ ID NO:64) and primer 2 (FL-SEQ ID NO:65), each 0.2 μ M D18S51 primer 1 (FL-SEQ ID NO:62) and primer 2 (SEQID NO:63), each 1.1 μ M HUMvWFA31 primers 1 (SEQ ID NO:76) and primer 2 (TMR-SEQ ID NO:40), each 1.8 μ M D8S1179 primers 1 (SEQ IDNO:74) and primer 2 (TMR-SEQ ID NO:75), each 0.6 μ M HUMTPOX primer 1 (SEQ ID NO:72) and primer 2 (TMR-SEQ ID NO:73), each 2.4 μ M HUMFIBRA primer 1 (TMR-SEQ ID NO:70) and primer 2 (SEQID NO:71), each 0.2 μ M D5S818 primer 1 (SEQ ID NO:84) and primer 2 (R6G-SEQ ID NO:85), each 0.1 μ M D13S317 primer 1 (SEQ ID NO:82) and primer 2 (R6G-SEQ ID NO:83), each 0.2 μ M D7S820 primer 1 (R6G-SEQ ID NO:80) and primer 2 (SEQ ID NO:81), each 0.15 μ MD16S539 primer 1 (SEQ ID NO:29) and primer 2 (R6G-SEQ ID NO:79), each 0.2 μ M HUMCSF1PO primer 1 (SEQ ID NO:77) and primer 2 (R6G-SEQ ID NO:78).
The product of amplification separates with ABI PRISM 310 genetic analyzers.DNA sample and 24 μ l application of sample solution (deionization methane amide) and the inner swimming lane size criteria of 1.0 μ l thing are mixed, 95 ℃ of sex change 3 minutes, and before injecting in precooling on ice.(Perkin Elmer Biosystems, Foster City CA) separate in 47cm * 50 μ m kapillaries with PerformanceOptimized Polymer 4 (POP-4).Use the producer's GeneScan to move (1ml) A of module GS STR POP4 (ibid).Deposition condition is injection in 5 seconds, and injection kV is 15.0, and electrophoresis kV is 15.0, and electrophoresis temperature is 60 ℃, and electrophoresis time is 28 minutes, and uses effective filter membrane A.
Figure 1A is the segmental scanning and printing result as the amplification of each locus of the above-mentioned ABI of using PRISM 310 genetic analyzers separation and detection.The locus D3S1358 of amplification when Figure 1A illustrates the DNA sample, HUMTH01, D21S11, D18S51, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, the amplified production of D16S539 and HUMCSF1PO.The peak is with fluorescein-labeled shown in the A group, and peak shown in the B group is with carboxyl tetramethyl-rhodamine mark, and the peak was with 5 shown in C organized, 6-carboxyl rhodamine 6G mark.
Figure 1B is the scanning and printing result as the sample of the sample same way as that scans among Figure 1A preparation, except in amplified reaction without dna profiling.Peak among this figure is to derive from dyestuff to put together background products with purification step and undefined reason.
With ABI PRISM 310 genetic analyzer fluoroscopic examination locus D3S1358,
HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, the multiplex amplification of HUMCSF1PO and S159
In this embodiment, each locus D3S1358 of the dna profiling that in a reaction tube, increases simultaneously, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO and S159.Pcr amplification is at 25 μ l, 1 * Gold ST*R damping fluid (50mM KCl, 10mM Tris-HCl (pH8.3 is at 25 ℃), 0.1%Triton X-100,1.5mM MgCl
2, 160 μ g/ml BSA and every kind of dATP of 200 μ M, dCTP, dGTP, and dTTP) in, with 1ng template and 4U AmpliTaq Gold
TMArchaeal dna polymerase carries out.(Perkin Elmer, Poster City use following amplification scheme in CA): 96 ℃ 12 minutes in GeneAmp PCR system 9600; Carried out 94 ℃ of 10 round-robin then 30 seconds, and in 68 seconds,, kept 30 seconds, in 50 seconds,, kept 45 seconds gradually to 70 ℃ gradually to 58 ℃; Carry out subsequently 20 round-robin 90 ℃ 30 seconds, in 60 seconds,, kept 30 seconds gradually to 58 ℃, in 50 seconds,, kept 45 seconds gradually to 70 ℃; Then carry out 1 time 60 ℃ 30 minutes.
Be used in combination 32 amplimers, comprise each 0.12 μ M D3S1358 primer 1 (SEQID NO:68) and primer 2 (FL-SEQ ID NO:69), each 0.08 μ M HUMTH01 primer 1 (FL-SEQ ID NO:66) and primer 2 (SEQ ID NO:67), each 0.8 μ M D21S11 primer 1 (SEQ ID NO:64) and primer 2 (FL-SEQ ID NO:65), each 0.2 μ M D18S51 primer 1 (FL-SEQ ID NO:62) and primer 2 (SEQID NO:63), each 0.24 μ M G475 primer 1 (FL-SEQ ID NO:88) and primer 2 (SEQ ID NO:89), each 0.6 μ M Amelogenin primer 1 (TMR-SEQID NO:86) and primer 2 (SEQ ID NO:87), each 1.1 μ M HUMvWFA31 primers 1 (SEQ ID NO:76) and primer 2 (TMR-SEQ ID NO:40), each 1.8 μ M D8S1179 primer 1 (SEQ ID NO:74) and primer 2 (TMR-SEQ IDNO:75), each 0.6 μ M HUMTPOX primer 1 (SEQ ID NO:72) and primer 2 (TMR-SEQ ID NO:73), each 2.4 μ M HUMFIBRA primers 1 (TMR-SEQ ID NO:70) and primer 2 (SEQ ID NO:71), each 0.2 μ M D5S818 primer 1 (SEQ ID NO:84) and primer 2 (R6G-SEQ ID NO:85), each 0.1 μ M D13S317 primer 1 (SEQ ID NO:82) and primer 2 (R6G-SEQ ID NO:83), each 0.2 μ M D7S820 primer 1 (R6G-SEQ ID NO:80) and primer 2 (SEQ ID NO:81), each 0.15 μ M D16S539 primer 1 (SEQ ID NO:29) and primer 2 (R6G-SEQ ID NO:79), each 0.2 μ M HUMCSF1PO primer 1 (SEQ ID NO:77) and primer 2 (R6G-SEQ ID NO:78), each 0.1 μ M S159 primer 1 (SEQ ID NO:90) and primer 2 (R6G-SEQ ID NO:91).
The product of amplification separates with ABI PRISM 310 genetic analyzers.DNA sample and 24 μ l application of sample solution (deionization methane amide) and the inner swimming lane size criteria of 1.0 μ l thing are mixed, 95 ℃ of sex change 3 minutes, and before injecting in precooling on ice.(Perkin Elmer Biosystems, Foster City CA) separate in 47cm * 50 μ m kapillaries with PerformanceOptimized Polymer 4 (POP-4).Use the producer's GeneScan to move (1ml) A of module GS STR POP4 (ibid).Deposition condition is injection in 5 seconds, and injection kV is 15.0, and electrophoresis kV is 15.0, and electrophoresis temperature is 60 ℃, and electrophoresis time is 28 minutes, and uses effective filter membrane A.
Fig. 2 A is the segmental scanning and printing result as the amplification of each locus of the above-mentioned ABI of using PRISM 310 genetic analyzers separation and detection.The locus D3S1358 of amplification when Fig. 2 A illustrates the DNA sample, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, the amplified production of HUMCSF1PO and S159.The peak is with fluorescein-labeled shown in the A group, and peak shown in the B group is with carboxyl tetramethyl-rhodamine mark, and the peak was with 5 shown in C organized, 6-carboxyl rhodamine 6G mark.
Fig. 2 B is the scanning and printing result as the sample of the sample same way as that scans among Fig. 2 A preparation, except in amplified reaction without dna profiling.Peak among this figure is to derive from dyestuff to put together background products with purification step and undefined reason.
With ABI PRISM 377DNA sequenator fluoroscopic examination locus D3S1358,
HUMTH01, D21S11, D18S51, G475, Ameloeenin, HUMvWFA31,
D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, the multiplex amplification of HUMCSF1PO and S159
In this embodiment, DNA sample such as embodiment 2 amplifications.The product of amplification separates with ABIPRISM 377DNA sequenator.This is with the thick 5%Long Ranger of 0.2mm
TM(ME), the 7M urea gel carries out acrylamide for FMC BioProducts, Rockland.The inner swimming lane size criteria of DNA sample and 1.5 μ l sample-loading buffers (88.25% methane amide, 4.1mM EDTA, 15mg/ml blue dextran) and 0.5 μ l thing mixes, 90 ℃ of sex change 2 minutes, and before application of sample in precooling on ice.GeneScan prerunning module (PRGS 36A-2400) and electrophoresis module (GS 36A-2400) with the producer carry out electrophoresis.Electrophoresis time 3 hours also uses effective filter membrane A.
Fig. 3 A is the segmental scanning and printing result as the amplification of each locus of the above-mentioned ABI of using PRISM 377 dna sequencing instrument separation and detection.The locus D3S1358 of amplification when Fig. 3 A illustrates the DNA sample, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, the amplified production of HUMCSF1PO and S159.The peak is with fluorescein-labeled shown in the A group, and peak shown in the B group is with carboxyl tetramethyl-rhodamine mark, and the peak was with 5 shown in C organized, 6-carboxyl rhodamine 6G mark.
Fig. 3 B is the scanning and printing result as the sample of the sample same way as that scans among Fig. 3 A preparation, except in amplified reaction without dna profiling.Peak among this figure is to derive from dyestuff to put together background products with purification step and undefined reason.
Embodiment 4
With Hitachi FMBIO II fluorescent scanning instrument fluoroscopic examination locus D3S1358,
HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, the multiplex amplification of HUMCSF1PO and S159
In this embodiment, two dna profilings all are being selected from locus D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, each locus combination of three different genes seat combinations of HUMCSF1PO and S159 is amplified simultaneously.The amplification of each locus combination comprises uses the 5ng template, is containing 25 μ l, 1 * Gold ST*R damping fluid (50mM KCl, 10mM Tris-HCl (pH8.3 is at 25 ℃), 0.1%Triton X-100,1.5mM MgCl
2, 160 μ g/ml BSA and every kind of dATP of 200 μ M, dCTP, dGTP, and dTTP) a reaction tube in carry out.
(Perkin Elmer, Foster City CA) are used for following amplification scheme: 96 ℃ 12 minutes in GeneAmp PCR system 9600; Carried out 94 ℃ of 10 round-robin then 30 seconds, and in 68 seconds,, kept 30 seconds, in 50 seconds,, kept 45 seconds gradually to 70 ℃ gradually to 58 ℃; Carry out subsequently 22 round-robin 90 ℃ 30 seconds, in 60 seconds,, kept 30 seconds gradually to 58 ℃, in 50 seconds,, kept 45 seconds gradually to 70 ℃; Then carry out 1 time 60 ℃ 30 minutes.
Use 32 amplimers of following concentration: comprise 0.225 μ M D3S1358 primer 1 (SEQ ID NO:68) and primer 2 (FL-SEQ ID NO:69), each 0.2 μ MHUMTH01 primer 1 (FL-SEQ ID NO:66) and primer 2 (SEQ ID NO:67), each 1.0 μ M D21S11 primers 1 (SEQ ID NO:64) and primer 2 (FL-SEQ ID NO:65), each 1.0 μ M D18S51 primers 1 (FL-SEQ ID NO:62) and primer 2 (SEQ ID NO:63), each 2.8 μ M G475 primers 1 (FL-SEQ ID NO:88) and primer 2 (SEQ ID NO:89), each 0.2 μ M Amelogenin primer 1 (TMR-SEQ ID NO:86) and primer 2 (SEQ ID NO:87), each 0.3 μ MHUMvWFA31 primer 1 (SEQ ID NO:76) and primer 2 (TMR-SEQ ID NO:40), each 1.5 μ M D8S1179 primers 1 (SEQ ID NO:74) and primer 2 (TMR-SEQ ID NO:75), each 0.2 μ M HUMTPOX primer 1 (SEQ ID NO:72) and primer 2 (TMR-SEQ ID NO:73), each 2.0 μ M HUMFIBRA primers 1 (TMR-SEQ ID NO:70) and primer 2 (SEQ ID NO:71), each 0.55 μ MD5S818 primer 1 (SEQ ID NO:84) and primer 2 (FL-SEQ ID NO:85), each 1.1 μ M D13S317 primer 1 (SEQ ID NO:82) and primer 2 (FL-SEQ IDNO:83), each 1.7 μ M D7S820 primers 1 (FL-SEQ ID NO:80) and primer 2 (SEQ ID NO:81), each 3.3 μ M D16S539 primers 1 (SEQ ID NO:29) and primer 2 (FL-SEQ ID NO:79), each 0.5 μ M HUMCSF1PO primer 1 (SEQID NO:77) and primer 2 (FL-SEQ ID NO:78), each 2.0 μ M S159 primers 1 (SEQ ID NO:90) and primer 2 (FL-SEQ ID NO:91).
In first locus combination, each template AmpliTaq Gold of 2.5U
TMArchaeal dna polymerase and above-mentioned concentration at each locus D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, the primer of HUMTPOX and HUMFIBRA increases.In second locus combination, the AmpliTaq Gold of 32 primers of all of above-mentioned concentration and 4U
TMArchaeal dna polymerase is used at all 16 locus D3S1358 of reaction tube amplification, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO and S159.In the 3rd locus combination, each template AmpliTaq Gold of 1.5U
TMArchaeal dna polymerase and above-mentioned concentration at each locus D5S818, D7S820, D13S317, D16S539, the primer of HUMCSF1PO and S159 increases.
0.4mm thick 4% denaturing polyacrylamide gel (acrylamide of 19: 1 ratio and bisacrylamide) electrophoretic separation (the Sam brook etc. of amplified production by containing 7M urea, (1989)), this gel and 2 glass plate chemically crosslinked (Kobayashi, Y. (1988), BRL Focus 10:73-74).DNA sample and 3.5 μ l application of sample solution (10mM NaOH, 95% methane amide, 0.05% bromjophenol blue) and the inner swimming lane size criteria of 0.5 μ l thing are mixed, 95 ℃ of sex change 2 minutes, and before application of sample in precooling on ice.Isolating product is by (Hitachi Software Engineering America, Ltd.South San Francisco CA) detect fluorescent signal and observe with HitachiFMIBO II fluorescent scanning instrument.505nm and 585nm bandpass filter are respectively applied for the locus that detects fluorescein-labeled locus and carboxyl tetramethyl-rhodamine mark.650nm bar bandpass filter is used to detect inner swimming lane size criteria thing (size criteria thing data not shown goes out).
Fig. 4 A and 4B have shown the fragment that derives from each amplified reaction.Fig. 4 A illustrates and derives from 505nm scanning result (fluorescein passage), and Fig. 4 B illustrates the 585nm scanning result (carboxyl tetramethyl-rhodamine passage) that derives from identical polypropylene acid amides swimming lane.For each dna profiling, swimming lane 1 illustrates amplification gene seat D3S1358 simultaneously, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, the result of the DNA sample of HUMTPOX and HUMFIBRA.Swimming lane 2 illustrates amplification gene seat D3S1358 simultaneously, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D13S317, D7S820, D16S539, the result of the DNA sample of HUMCSF1PO and S159.Swimming lane 3 illustrates amplification gene seat D5S818 simultaneously, D13S317, D7S820, D16S539, the result of the DNA sample of HUMCSF1PO and S159.
Embodiment 5
With Hitachi FMBIO II fluorescent scanning instrument fluoroscopic examination locus D3S1358,
HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, the multiplex amplification of HUMCSF1PO and S159
In this embodiment, two dna profilings all are being selected from locus D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, each locus combination of two different genes seat combinations of HUMCSF1PO and S159 is amplified simultaneously.The amplification of each locus combination comprises uses the 5ng template, is containing 25 μ l, 1 * Gold ST*R damping fluid (50mM KCl, 10mM Tris-HCl (pH8.3 is at 25 ℃), 0.1% Triton X-100,1.5mM MgCl
2, 160 μ g/ml BSA and every kind of dATP of 200 μ M, dCTP, dGTP, and dTTP) a reaction tube in carry out.
(Perkin Elmer, Foster City CA) are used for following amplification scheme: 96 ℃ 12 minutes in GeneAmp PCR system 9600; Carried out 94 ℃ of 10 round-robin then 30 seconds, and in 68 seconds,, kept 30 seconds, in 50 seconds,, kept 45 seconds gradually to 70 ℃ gradually to 58 ℃; Carry out subsequently 22 round-robin 90 ℃ 30 seconds, in 60 seconds,, kept 30 seconds gradually to 58 ℃, in 50 seconds,, kept 45 seconds gradually to 70 ℃; Then carry out 1 time 60 ℃ 30 minutes.
Use 32 amplimers of following concentration: comprise 0.225 μ M D3S1358 primer 1 (SEQ ID NO:68) and primer 2 (FL-SEQ ID NO:69), each 0.2 μ MHUMTH01 primer 1 (FL-SEQ ID NO:66) and primer 2 (SEQ ID NO:67), each 1.0 μ M D21S11 primers 1 (SEQ ID NO:64) and primer 2 (FL-SEQ ID NO:65), each 1.0 μ M D18S51 primers 1 (FL-SEQ ID NO:62) and primer 2 (SEQ ID NO:63), each 2.8 μ M G475 primers 1 (SEQ ID NO:88) and primer 2 (FL-SEQ ID NO:94), each 0.2 μ M Amelogenin primer 1 (TMR-SEQ ID NO:86) and primer 2 (SEQ ID NO:87), each 0.3 μ M HUMvWFA31 primer 1 (SEQ ID NO:76) and primer 2 (TMR-SEQ IDNO:40), each 1.5 μ M D8S1179 primers 1 (SEQ ID NO:74) and primer 2 (TMR-SEQ ID NO:75), each 0.2 μ M HUMTPOX primer 1 (SEQ IDNO:72) and primer 2 (TMR-SEQ ID NO:73), each 2.0 μ M HUMFIBRA primers 1 (TMR-SEQ ID NO:70) and primer 2 (SEQ ID NO:71), each 0.55 μ M D5S818 primer 1 (SEQ ID NO:84) and primer 2 (FL-SEQ ID NO:85), each 1.1 μ M D13S317 primers 1 (SEQ ID NO:82) and primer 2 (FL-SEQ ID NO:83), each 1.7 μ M D7S820 primers 1 (FL-SEQ ID NO:80) and primer 2 (SEQ ID NO:81), each 3.3 μ M D16S539 primers 1 (SEQ IDNO:29) and primer 2 (FL-SEQ ID NO:79), each 0.5 μ M HUMCSF1PO primer 1 (SEQ ID NO:77) and primer 2 (FL-SEQ ID NO:78), each 2.0 μ M S159 primers 1 (SEQ ID NO:95) and primer 2 (FL-SEQ ID NO:96).
In first locus combination, each template AmpliTaq Gold of 2.5U
TMArchaeal dna polymerase and above-mentioned concentration at each locus D3S1358, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, the primer of HUMTPOX and HUMFIBRA increases.In second locus combination, the AmpliTaq Gold of 32 primers of all of above-mentioned concentration and 4U
TMArchaeal dna polymerase is used at all 16 locus D3S1358 of reaction tube amplification, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO and S159.
Amplified production is as separation and observation as described in the embodiment 4.
Fig. 5 A and 5B have shown the fragment that derives from each amplified reaction.Fig. 5 A illustrates and derives from 505nm scanning result (fluorescein passage), and Fig. 5 B illustrates the 585nm scanning result (carboxyl tetramethyl-rhodamine passage) that derives from identical polypropylene acid amides swimming lane.For each dna profiling, swimming lane 1 illustrates amplification gene seat D3S1358 simultaneously, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, the result of the DNA sample of HUMTPOX and HUMFIBRA.Swimming lane 2 illustrates amplification gene seat D3S1358 simultaneously, HUMTH01, D21S11, D18S51, G475, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D13S317, D7S820, D16S539, the result of the DNA sample of HUMCSF1PO and S159.
Embodiment 6
With Hitachi FMBIO II fluorescent scanning instrument fluoroscopic examination locus D3S1358,
HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31,
D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317,
D16S539, the multiplex amplification of HUMCSF1PO and C221
In this embodiment, two dna profilings all are being selected from locus D3S1358, HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, each locus combination of three different genes seat combinations of HUMCSF1PO and C221 is amplified simultaneously.The amplification of each locus combination comprises uses the 10ng template, is containing 25 μ l, 1 * Gold ST*R damping fluid (50mM KCl, 10mM Tris-HCl (pH8.3 is at 25 ℃), 0.1%Triton X-100,1.5mM MgCl
2, 160 μ g/ml BSA and every kind of dATP of 200 μ M, dCTP, dGTP, and dTTP) a reaction tube in carry out.
(Perkin Elmer, Foster City CA) are used for following amplification scheme: 96 ℃ 12 minutes in GeneAmp PCR system 9600; Carried out 94 ℃ of 10 round-robin then 30 seconds, and in 68 seconds,, kept 30 seconds, in 50 seconds,, kept 45 seconds gradually to 70 ℃ gradually to 60 ℃; Carry out subsequently 20 round-robin 90 ℃ 30 seconds, in 60 seconds,, kept 30 seconds gradually to 60 ℃, in 50 seconds,, kept 45 seconds gradually to 70 ℃; Then carry out 1 time 60 ℃ 30 minutes.
Use 32 amplimers of following concentration: comprise 0.75 μ M D3S1358 primer 1 (SEQ ID NO:106) and primer 2 (FL-SEQ ID NO:69), each 0.3 μ MHUMTH01 primer 1 (FL-SEQ ID NO:38) and primer 2 (SEQ ID NO:103), each 2.0 μ M D21S11 primers 1 (SEQ ID NO:64) and primer 2 (FL-SEQ ID NO:65), each 0.3 μ M D18S51 primer 1 (FL-SEQ ID NO:101) and primer 2 (SEQ ID NO:102), each 2.0 μ M S159 primers 1 (SEQ ID NO:92) and primer 2 (FL-SEQ ID NO:93), each 0.15 μ M Amelogenin primer 1 (TMR-SEQ ID NO:105) and primer 2 (SEQ ID NO:87), each 1.0 μ MHUMvWFA31 primers 1 (SEQ ID NO:76) and primer 2 (TMR-SEQ IDNO:40), each 1.25 μ M D8S1179 primers 1 (TMR-SEQ ID NO:104) and primer 2 (SEQ ID NO:75), each 0.75 μ M HUMTPOX primer 1 (TMR-SEQ ID NO:72) and primer 2 (SEQ ID NO:73), each 1.5 μ M HUMFIBRA primers 1 (TMR-SEQ ID NO:70) and primer 2 (SEQ ID NO:107), each 0.55 μ M D5S818 primer 1 (SEQ ID NO:84) and primer 2 (FL-SEQ ID NO:85), each 1.1 μ M D13S317 primers 1 (SEQ ID NO:3) and primer 2 (FL-SEQ ID NO:4), each 1.7 μ M D7S820 primers 1 (FL-SEQ ID NO:80) and primer 2 (SEQ ID NO:81), each 3.3 μ M D16S539 primers 1 (FL-SEQ IDNO:29) and primer 2 (SEQ ID NO:97), each 0.25 μ M HUMCSF1PO primer 1 (SEQ ID NO:77) and primer 2 (FL-SEQ ID NO:98), each 1.0 μ M C221 primers 1 (FL-SEQ ID NO:99) and primer 2 (SEQ ID NO:100).
In first locus combination, each template AmpliTaq Gold of 2.5U
TMArchaeal dna polymerase and above-mentioned concentration at each locus D3S1358, HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179, the primer of HUMTPOX and HUMFIBRA increases.In second locus combination, the AmpliTaq Gold of 32 primers of all of above-mentioned concentration and 4U
TMArchaeal dna polymerase is used at all 16 locus D3S1358 of reaction tube amplification, HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D7S820, D13S317, D16S539, HUMCSF1PO and C221.In the 3rd locus combination, each template AmpliTaq Gold of 1.5U
TMArchaeal dna polymerase and above-mentioned concentration at each locus D5S818, D7S820, D13S317, D16S539, the primer of HUMCSF1PO and C221 increases.
Amplified production is as separation and detection as described in the embodiment 4, except with each sample of amplified production with 1 * STR damping fluid (50mM KCl, 10mM Tris-HCl (pH8.3 is at 25 ℃), 0.1% Triton X-100,1.5mM MgCl
2With every kind of dATP of 200 μ M, dCTP, dGTP and dTTP) with dilution in 1: 4.The application of sample solution (10mM NaOH, 95% methane amide, 0.05% bromjophenol blue) that amplified production (2.5 μ l) and the 2.5 μ l of dilution is not contained inner swimming lane standard substance mixes, 95% sex change 2 minutes, and before application of sample in precooling on ice.
Fig. 6 A and 6B have shown the fragment that derives from each amplified reaction.Fig. 6 A illustrates and derives from 505nm scanning result (fluorescein passage), and Fig. 6 B illustrates the 585nm scanning result (carboxyl tetramethyl-rhodamine passage) that derives from identical polypropylene acid amides swimming lane.For each dna profiling, swimming lane 1 illustrates amplification gene seat D3S1358 simultaneously, HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179, the result of the DNA sample of HUMTPOX and HUMFIBRA.Swimming lane 2 illustrates amplification gene seat D3S1358 simultaneously, HUMTH01, D21S11, D18S51, S159, Amelogenin, HUMvWFA31, D8S1179, HUMTPOX, HUMFIBRA, D5S818, D13S317, D7S820, D16S539, the result of the DNA sample of HUMCSF1PO and C221.Swimming lane 3 illustrates amplification gene seat D5S818 simultaneously, D13S317, D7S820, D16S539, the result of the DNA sample of HUMCSF1PO and C221.
Sequence table
Claims (27)
1. the allelic method that exists in definite simultaneously series of genes seat from one or more DNA sample comprises:
(a) the allelic mixture that this serial genes seat in the described DNA sample of coamplification increases with generation in a multiplex amplification reaction, wherein said locus comprises D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA and HUMvWFA31; With
(b) estimate the allelotrope of the amplification in the mixture, with determine in the DNA sample should each analysis of series the allelotrope that exists of locus.
2. the method for claim 1 also comprises at least 1 short tandem repeat that is selected from next group: D3S1539, D4S2368, D9S930, D10S1239, D14S118, D14S548, D14S562, D16S490, D16S753, D17S1298, D17S1299, D19S253, D20S481, D22S683, HUMF13A01, HUMBFXIII, HUMLIPOL.
3. the process of claim 1 wherein that this serial genes seat also comprises the pentanucleotide tandem repeat.
4. the method for claim 3, wherein the pentanucleotide tandem repeat is selected from G475, C221 and S159.
5. the process of claim 1 wherein that this serial genes seat also comprises the locus of the sex at least one source that can be used to differentiate the DNA sample.
6. the method for claim 5, wherein at least one source of DNA sample is the people, and is used to differentiate that other locus of human nature is the Amelogenin locus.
7. the method for claim 6, wherein the Amelogenin locus is with having the SEQ of being selected from ID NO:86, the Oligonucleolide primers coamplification of the sequence of SEQ ID NO:87 and SEQ ID NO:105.
8. each method in the claim 1,2,3 or 5, wherein multiplex amplification reaction is with having the Oligonucleolide primers that is selected from following at least one group of sequence to carrying out, described group is:
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80 and SEQ ID NO:81 are when one of this serial genes seat is D7S820;
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82 and SEQ ID NO:83 are when one of this serial genes seat is D13S317;
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:84 and SEQ ID NO:85 are when one of this serial genes seat is D5S818;
SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:49 are when one of this serial genes seat is D3S1539;
SEQ ID NO:9, SEQ ID NO:10 is when one of this serial genes seat is D17S1298;
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:52, SEQ ID NO:53 is when one of this serial genes seat is D20S481;
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:55, SEQ ID NO:61 is when one of this serial genes seat is D9S930;
SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:54 is when one of this serial genes seat is D10S1239;
SEQ ID NO:17, SEQ ID NO:18 is when one of this serial genes seat is D14S118;
SEQ ID NO:19, SEQ ID NO:20 is when one of this serial genes seat is D14S562;
SEQ ID NO:21, SEQ ID NO:22 is when one of this serial genes seat is D14S548;
SEQ ID NO:23, SEQ ID NO:24 is when one of this serial genes seat is D16S490;
SEQ ID NO:25, SEQ ID NO:26 is when one of this serial genes seat is D16S753;
SEQ ID NO:27, SEQ ID NO:28 is when one of this serial genes seat is D17S1299;
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID NO:79 and SEQ ID NO:97 are when one of this serial genes seat is D16S539;
SEQ ID NO:31, SEQ ID NO:32 is when one of this serial genes seat is D22S683;
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:98 are when one of this serial genes seat is HUMCSF1PO;
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:76 are when one of this serial genes seat is HUMvWFA31;
SEQ ID NO:41, SEQ ID NO:42 is when one of this serial genes seat is HUMF13A01;
SEQ ID NO:45, SEQ ID NO:46 is when one of this serial genes seat is HUMBFXIII;
SEQ ID NO:47, SEQ ID NO:48 is when one of this serial genes seat is HUMLIPOL;
SEQ ID NO:50, SEQ ID NO:51 is when one of this serial genes seat is D19S253;
SEQ ID NO:56, SEQ ID NO:57 is when one of this serial genes seat is D4S2368;
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101 and SEQ IDNO:102 are when one of this serial genes seat is D18S51;
SEQ ID NO:64 and SEQ ID NO:65 are when one of this serial genes seat is D21S11;
SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:106 are when one of this serial genes seat is D3S1358;
SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:107 are when one of this serial genes seat is HUMFIBRA;
SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:104 are when one of this serial genes seat is D8S1179;
SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:105 are when one of this serial genes seat is Amelogenin;
SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:94 are when one of this serial genes seat is G475;
SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95 and SEQ ID NO:96 are when one of this serial genes seat is S159; With
SEQ ID NO:99 and SEQ ID NO:100 are when one of this serial genes seat is C221.
9. the process of claim 1 wherein that the allelotrope of amplification separated before the evaluation of step (c), the separate mode of use is selected from polyacrylamide gel electrophoresis and capillary gel electrophoresis.
10. the process of claim 1 wherein that multiplex amplification reaction is to use the Oligonucleolide primers of the locus both sides that are positioned at analysis to carrying out.
11. the method for claim 10, wherein this serial genes seat polymerase chain reaction coamplification.
12. the method for claim 10, wherein the locus of each coamplification in multiple reaction is with a pair of primer coamplification that is positioned at these locus both sides, wherein fluorescent mark with covalent attachment of at least one primer of every pair of primer.
13. the method for claim 12, wherein at least 3 primers in the primer of mark have the different fluorescent marks of covalent attachment.
14. the method for claim 1, wherein said at least one DNA specimen preparation to be analyzed is organized from the people, and wherein people tissue is selected from blood, seminal fluid, vaginal cell, hair, saliva, urine, contains amniotic fluid, and any mixture of above-mentioned tissue of placenta cells or fetal cell.
15. the process of claim 1 wherein the allelotrope that increases by itself and the contrast of size criteria thing are estimated, wherein the size criteria thing is selected from dna marker and locus-specific allelotrope ladder.
16. the process of claim 1 wherein that multiplex amplification reaction is to use at least one Oligonucleolide primers of each locus in this serial genes seat carrying out, wherein at least one primer has the sequence that is selected from following each group primer sequence:
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101 and SEQ IDNO:102 are when one of this serial genes seat is D18S51;
SEQ ID NO:64 and SEQ ID NO:65 are used for locus D21S11;
SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:106 are used for locus D3S1358;
SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:107 are used for locus HUMFIBRA;
SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:104 are used for locus D8S1179;
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:76 are used for locus HUMvWFA31;
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:98 are used for locus HUMCSF1PO;
SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:58, SEQ ID NO:79 and SEQ ID NO:97 are used for locus D16S539;
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:80 and SEQ ID NO:81 are used for locus D7S820;
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82 and SEQ ID NO:83 are used for locus D13S317; With
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:84 and SEQ ID NO:85 are used for locus D5S818.
17. the process of claim 1 wherein that this serial genes seat also comprises is selected from G475, S159, at least one extra locus of C221 and Amelogenin.
18. the method for claim 17, wherein multiplex amplification reaction is with having at least one Oligonucleolide primers of being selected from least with at least one primer sequence of next group sequence to carrying out:
SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:94 are when one of this serial genes seat is G475;
SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:95 and SEQ ID NO:96 are when one of this serial genes seat is S159;
SEQ ID NO:99 and SEQ ID NO:100 are when one of this serial genes seat is C221; With
SEQ ID NO:86, SEQ ID NO:87 and SEQ ID NO:105 are when one of this serial genes seat is the Amelogenin locus.
19. the process of claim 1 wherein that multiplex amplification reaction is a polymerase chain reaction.
20. the method for claim 1, at least one DNA specimen preparation wherein to be analyzed is organized from the people, and wherein people tissue is selected from blood, seminal fluid, vaginal cell, hair, saliva, urine, bone, cheek sample, contains amniotic fluid, and any mixture of above-mentioned tissue of placenta cells or fetal cell.
21. the test kit of the series of genes seat of a while analyzing gene group DNA, the Oligonucleolide primers that comprises the series of genes seat that is used for coamplification genomic dna to be analyzed, wherein this serial genes seat comprise following can be by the short tandem repeat of coamplification: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, HUMCSF1PO, HUMFIBRA and HUMvWFA31, and wherein primer is included in one or more container.
22. the test kit of claim 21, wherein all Oligonucleolide primers all are included in the container in the test kit.
23. the test kit of claim 21, wherein genomic dna is the human gene group DNA.
24. the test kit of claim 21, at least one the right primer of primer that wherein is used for a locus of this serial genes seat of coamplification has the sequence that is selected from next group:
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:101 and SEQ IDNO:102 are used for locus D18S51;
SEQ ID NO:64 and SEQ ID NO:65 are used for locus D21S11;
SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:106 are used for locus D3S1358;
SEQ ID NO:70, SEQ ID NO:71 and SEQ ID NO:107 are used for locus HUMFIBRA;
SEQ ID NO:74, SEQ ID NO:75 and SEQ ID NO:104 are used for locus D8S1179;
SEQ ID NO:76 and SEQ ID NO:40 are used for locus HUMvWFA31;
SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:98 are used for locus HUMCSF1PO;
SEQ ID NO:29, SEQ ID NO:79 and SEQ ID NO:97 are used for locus D16S539;
SEQ ID NO:80 and SEQ ID NO:81 are used for locus D7S820;
SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:82 and SEQ ID NO:83 are used for locus D13S317; With
SEQ ID NO:84 and SEQ ID NO:85 are used for locus D5S818.
25. the test kit of claim 21 also comprises the reagent that is used at least one multiplex amplification reaction.
26. the test kit of claim 21 also comprises the container with allelotrope ladder.
27. the test kit of claim 26, wherein each horizontal bar of allelotrope ladder and be used for the fluorescent mark that at least one Oligonucleolide primers of this each locus of series has covalent attachment, and at least 2 fluorescent marks of these Oligonucleolide primers with covalent attachment different with other primer in the container.
Applications Claiming Priority (2)
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| US09/199,542 | 1998-11-25 | ||
| US09/199,542 US6479235B1 (en) | 1994-09-30 | 1998-11-25 | Multiplex amplification of short tandem repeat loci |
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| CNB998137294A Division CN100366753C (en) | 1998-11-25 | 1999-11-24 | Multiplex amplification of short tandem repeat loci |
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| CNB998137294A Expired - Lifetime CN100366753C (en) | 1998-11-25 | 1999-11-24 | Multiplex amplification of short tandem repeat loci |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB998137294A Expired - Lifetime CN100366753C (en) | 1998-11-25 | 1999-11-24 | Multiplex amplification of short tandem repeat loci |
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| US (1) | US6479235B1 (en) |
| EP (1) | EP1135530B1 (en) |
| JP (2) | JP5456950B2 (en) |
| CN (2) | CN101230394A (en) |
| AU (1) | AU774626B2 (en) |
| BR (1) | BR9915597A (en) |
| CA (1) | CA2352042A1 (en) |
| ES (1) | ES2394070T3 (en) |
| PT (1) | PT1135530E (en) |
| WO (1) | WO2000031306A2 (en) |
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| CN103917661A (en) * | 2011-05-12 | 2014-07-09 | 网络百奥有限公司 | Methods and compositions for rapid multiplex amplification of STR loci |
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| JP2010081944A (en) | 2010-04-15 |
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| JP5711887B2 (en) | 2015-05-07 |
| BR9915597A (en) | 2001-10-23 |
| EP1135530B1 (en) | 2012-09-19 |
| WO2000031306A2 (en) | 2000-06-02 |
| CN1332805A (en) | 2002-01-23 |
| PT1135530E (en) | 2012-12-18 |
| WO2000031306A9 (en) | 2002-08-29 |
| US6479235B1 (en) | 2002-11-12 |
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